Class I MHC Ag cross processing allows exogenous or vacuolar Ags to be processed for presentation to CD8+ T cells. Cross processing is essential for priming of CD8+ naive T cells by dendritic cells (DCs) in lymph nodes. A less recognized but potentially critical role of cross processing is to allow cells in non-lymphoid organs that are infected with vacuolar pathogens, e.g. Mycobacterium tuberculosis (MTB), to present pathogen-derived antigens to CD8+ effector T cells to elicit cytokine production or cytolytic function. DCs and macrophages may both harbor MTB and may hypothetically present MTB Ags to CD8+ effector T cells, allowing CD8+ T cells to contribute to containment of MTB infection. Research direction: This grant is to study basic mechanisms of bacterial Ag processing with a primary focus on MHC-I cross processing. MTB is selected as a model organism that represents an excellent model for processing of intravacuolar pathogens in addition to its significance as a human pathogen. Questions to be solved: We do not know which MTB-infected APCs have cross processing function; this has important implications for generation of immune responses vs. immune evasion. We do not understand mechanisms for cross processing of vacuolar pathogens, e.g. MTB, and cross processing functions of physiologically important lung APCs have not been investigated. Hypothesis: Cross processing of vacuolar organisms (e.g. MTB) allows recognition of infected cells by CD8+ T cells, contributing to host defense. DCs and macrophages may both cross present Ags to effector T cells, although they may use different cross processing mechanisms, undergo different regulation by pathogens, and play different roles in immune responses. Aim 1 addresses basic mechanisms of cross processing of MTB. Aim 2 investigates the cross processing functions of lung APCs. Aim 3 addresses regulation of cross processing by Toll-like receptors, interferons and MTB. Significance in lay terms: These studies will discover mechanisms that allow immune recognition of the causative agent of tuberculosis by CD8+ T cells, which help fight tuberculosis infection. This will increase our understanding of tuberculosis pathogenesis and aid development of strategies for enhanced vaccine design or immunotherapy for tuberculosis and other diseases.